Abstract The physical properties of the cell interior are crucial for the organization and efficiency of biochemical reactions. The biophysical properties of cells can change during development, cancer progression and aging. Thus, it is crucial to understand the mechanisms that control the physical properties of the cell and the physiological consequences of perturbations to this unique environment. The current gold-standard method to study properties of the cell interior is the microinjection of inorganic nanoparticles. This technique dilutes the cytoplasm, damages the membrane and cortex, and is highly prone to experimental error. Microinjection is impossible in key genetic systems such as S. cerevisiae and has been mostly limited to cell culture models due to the difficulty of applying current techniques to animals. Studies of organelles have been almost impossible, limiting our current understanding to the cytoplasm. Finally, microinjection is labor intensive, making it impossible to undertake large- scale genetic screens to find genes and pathways that control the properties of the cell interior. We have created self-assembling, genetically-encoded fluorescent probes (GEMs) with 20- and 40-nm diameters that overcome all of the problems of the previous state-of-the-art technologies. After inserting the gene encoding GEMs, cells have nanoparticles permanently present, thus no microinjection is required. GEMs massively increase the speed, efficiency and reproducibility of microrheology experiments. Our recent discovery of pathways that control the physical properties of the cytoplasm required hundreds of experiments in an extensive genetic screen, which would not have been feasible without GEMs. We will use this focused technology research funding to extend the GEM technology and make it accessible to a broad community of scientists. In Aim 1, we will target GEMs to the nucleus and mitochondria, to characterize these organelles for the first time. In Aim 2, we will generate nanoparticles from 50 nm to 100 nm in size. The cellular environment varies substantially for objects of different sizes, just as car and a bicycle experience a traffic jam differently. Thus, we must investigate the environment for a wide range of particle sizes. Finally, in Aim 3, we will extend GEM technology to animals with well-defined developmental patterns to enable characterization of the physical properties of cells within tissues throughout development. Throughout, we will develop computational tools to identify and track GEMs, compare our technology to current gold-standard techniques and generate reference datasets that will aid the community in future studies. Overall, we will develop a suite of easy-to-use nanoparticles that will accelerate the discovery of mechanisms that control the physical properties of animals, cells and organelles. This will help elucidate the role of the intracellular environment to cell function, and the contributions of the loss of this physical homeostasis to disease.